1. Field of the Invention
The present invention relates to a novel method for synthesis, isolation and purification of peptides. More particularly, the invention relates to penI fusion polypeptides, peptide synthesis, isolation and purification methods that comprise use of penI fusion polypeptides, and related gene fusion constructs that code for such penI fusion polypeptides.
2. Background
Recombinant DNA methods are employed to produce a variety of peptides. Recombinant DNA technology includes expression of a gene, either synthesized or isolated, to produce a peptide of interest. In brief, a desired DNA sequence is ligated into a cloning vector such as a plasmid. A host cell such as E. coli, is transformed with the cloning vector and the transformed host is then cultivated under conditions suitable for expression of the peptide coded for by the DNA sequence. The thus produced proteins are then isolated from the culture medium and typically must be purified. For example, the expressed peptide is often secreted by the host cells into the culture medium and the peptide must be separated from the culture medium and other material in that medium such as cell waste products, other peptides, etc.
Peptides also may be produced by chemical synthesis techniques, although in many applications a chemical synthetic approach may be less preferred than a recombinant procedure. For example, peptides greater than about forty or fifty amino acids in length often cannot be chemically synthesized in acceptable yields. Further, purification of relatively large chemically synthesized peptides often can be more burdensome than corresponding recombinant peptides.
A number of methods have been reported to isolate and purify proteins, including peptides produced by chemical synthesis or recombinant DNA techniques. For example, known purification methods include centrifugation, column chromatography and electrophoresis. While these methods can produce a purified peptide, they each require one or more additional and often burdensome purification steps after initial purification of the peptide. Moreover, in many current isolation and purification procedures, a significant amount of the crude peptide is lost during the procedure resulting in reduced yields.
In certain prior peptide isolation and purification schemes, hybrid or fusion polypeptides have been employed. These approaches have generally provided construction of a gene fusion that codes for a polypeptide that contains a peptide of interest linked to a peptide that exhibits specific binding characteristics not exhibited by the peptide of interest.
These prior fusion peptide methods present notable shortcomings including low yields of purified protein as well as multiple step isolation and purification protocols. For example, in EP 0244147 a fusion polypeptide is described that contains a beta-galactosidase moiety linked to a desired peptide through a renin cleavage site. To isolate and purify the desired peptide, the fusion polypeptide is adsorbed to an affinity matrix and, after eluting to remove other proteins, renin is added to the bound complex to cleave the fusion polypeptide and release the peptide of interest while the beta-galactosidase remains bound to the matrix. In a still further step the isolated peptide is purified by anion exchange chromatography.
It thus would be desirable to have new and simple methods for the synthesis and purification of peptides, particularly peptides produced by recombinant means. It would be further desirable to have such new methods that enable convenient isolation and purification of a desired peptide in a single step, particularly without significant reduction in yields.